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Fleay had an aesthetic upbringing: His mother, Maude Glover Fleay, had studied painting under Fred McCubbin; his father, William Henry Fleay, was a manufacturing chemist in Ballarat. After education at a state primary school and later a private high school, Ballarat Grammar School, Fleay was first employed in his fathers chemist shop and then was briefly a teacher at Ballarat Grammar.

He left for Melbourne in 1927 to study for a Bachelor of Science degree and Diploma of Education at Melbourne University. There, he met another student, Mary Sigrid Collie, and they married in 1931, the same year that Fleay graduated having majored in zoology, botany and education. He was employed as a teacher in Ballarat until 1934.

Fleay's interest in the natural world coincided with the awakening of scientific interest in endangered species, and the realisation by the public that Australian animals were worthy of attention other than as a source of food.

He realised the importance of endangered species early in his career when, in 1933, he was the last person to photograph a captive thylacine or Tasmanian tiger at the Hobart Zoo. In the process he was bitten on the buttocks, the scar from the injury carried proudly throughout his life.

In 1934, Fleay was asked to design and establish the Australian animal section at Melbourne Zoo, and worked there for 4 years. During this time he had several scientific achievements, including the first breeding in captivity of the emu, several bird species including the tawny frogmouth, and marsupials including the koala. He also commenced research into the breeding habits of the platypus. His next public education efforts were nature talks on a Melbourne radio station, in 1937. Later that year, disagreements with the zoo's management came to a head and Fleay was dismissed, principally because of his belief that native birds and animals should be fed what they would eat in the wild.

Coincidentally, the Healesville Sanctuary some 90 km from Melbourne was in need of a director and he was appointed. The animals there included quolls, Tasmanian devils, dingoes and various birds of prey, to which he added tiger snakes that were milked for antivenene, and platypus. Many animals were housed in large paddock-like areas with swing-weighted gates so that visitors could freely interact with the animals. He also conducted daily 'feeding' displays of the platypuses.

His greatest achievement at Healesville was in 1943, when he bred the first platypus in captivity. His platypusary (platypus enclosure) incorporated features of their native streams. On about 5 November 1943, "Corrie" was born. No-one other than Fleay successfully bred and reared a platypus until 1998 when Healseville Sanctuary again had success. Since then, breeding has occurred only twice more: at Healesville and Taronga Zoo (twins).

Between 1945–1947, Fleay led an expedition to Tasmania in an attempt to capture a breeding pair of thylacines, however he returned empty handed.[1]

In 1947, he took three platypuses to New York for the Bronx Zoo where they occupied a platypusary built to his specifications. He studied animal husbandry at various zoos and wildlife sanctuaries across the US, returning to Healesville in October to discover that the Board had dismissed him for supposed unauthorised donations of animals to various foreign zoos. While untrue, this caused considerable hurt: He was demoted and remained at Healesville as a consultant.

He was also keeping a private collection of animals, but in 1951 the Government of Victoria legislated to prevent private individuals from charging fees for the public to see animal collections. This caused him to decide to move the collection.

Map showing past and present distribution of the bridled nailtail wallaby. This map is an inset from a sign located at David Fleay Wildlife Park in Burleigh Heads, Queensland.

After extensive research, the Tallebudgera Estuary in the hinterland behind Burleigh on the Gold Coast in Queensland was selected, the reasons including that it offered an untouched natural habitat for koalas apart from already having cleared areas (then farmland) for development of animal enclosures. The Fleays gradually acquired land and by 1958 had enclosures for people to see platypuses, snakes, dingoes, plain turkeys, ospreys, crocodiles and alligators; in contrast, bandicoots, flying foxes, sea eagles, wallabies and koalas, were free to visit from adjoining the forest. However, his focus was on the scientific study of the animals.

The area also included middens used by earlier generations of the Gold Coast's Kombumerri Aboriginal people. Fleay retained these heritage areas, and maintained good relationships with the Kombumerri.

The animals were fed partly from donations from local bakers and butchers, with local residents donating dead animals to feed the owls (or the goannas if no longer fresh); mice and rats were collected frequently from the McKerras Research Institute behind the hospital; worms were collected fresh daily for the platypuses; eels, pigeons and flying foxes were also killed to provide food for the owls, snakes and crocodiles.

Injured or sick animals from as far away as New Guinea and Central Queensland were accommodated at the sanctuary. Those that lived were kept for research or breeding; native animals, when recovered, were released into the wild; deceased animals were fed to the survivors.

In 1982, 37 acres (150,000 m²) of the land owned by David and Sigrid Fleay was sold to the Queensland Government and became a Conservation Park. The following year, the 20 acre (81,000 m²) main Fauna Reserve with its animal enclosures was also sold to the Government. The remainder of the site 7.5 acres (30,000 m²) was transferred in 1985. Under the terms of this arrangement, David and Sigrid Fleay continued to live and work at the park: In 1983 it closed for 5 years for redevelopment and re-opened in 1988. The government retains the property as the David Fleay Wildlife Park.